The sessile nature of plant life generates a constant exposure to environmental factors that exert positive and negative effects on its growth and development. One of the major impediments facing modem agriculture is adverse environmental conditions. One important factor which causes significant crop loss is heat stress. Temperature stress greatly reduces grain yield in many cereal crops such as maize, wheat, and barley. Yield decreases due to heat stress range from 7 to 35% in the cereals of world-wide importance.
A number of studies have identified likely physiological consequences of heat stress. Early work by Hunter et al. (1977) using growth chamber conditions showed that temperature decreased the duration of grain filling in maize. Similar results in which the duration of grain filling was adversely altered by increased temperatures were identified by Tollenaar and Bruulsema (1988). Badu-Apraku et al. (1983) measured a marked reduction in the yield of maize plants grown under the day/night temperature regime of 35/15° C. compared to growth in a 25/15° C. temperature regime. Reduced yields due to increased temperatures is also supported by historical as well as climatological studies (Thompson 1986; Thompson 1975; Chang 1981; Conroy et al., 1994). That the physiological processes of the developing seed are adversely affected by heat stress is evident from studies using an in vitro kernel culture system (Jones et al., 1981; Jones et al., 1984; Cheikh et al., 1995). Maize kernels cultured at the above-optimum temperature of 35° C. exhibited a dramatic reduction in weight.
Work with wheat identified the loss of soluble starch synthase (SSS) activity as a hallmark of the wheat endosperm's response to heat stress (Hawker et al., 1993; Denyer et al., 1994; Jenner 1994). Additional studies with SSS of wheat endosperm show that it is heat labile (Rijven 1986; Keeling et al., 1993; Jenner et al., 1995).
ADP glucose pyrophosphorylase (AGP) is another important starch biosynthesis enzyme in plants. AGP catalyzes the conversion of ATP and α-glucose-1-phosphate to ADP-glucose and pyrophosphate. ADP-glucose is used as a glycosyl donor in starch biosynthesis by plants and in glycogen biosynthesis by bacteria. The importance of ADP-glucose pyrophosphorylase as a key enzyme in the regulation of starch biosynthesis was noted in the study of starch deficient mutants of maize (Zea mays) endosperm (Tsai et al., 1966; Dickinson et al., 1969). Biochemical and genetic evidence has identified AGP as a key enzyme in starch biosynthesis in higher plants and glycogen biosynthesis in E. coli (Preiss et al., 1994; Preiss et al., 1996). AGP catalyzes what is viewed as the initial step in the starch biosynthetic pathway with the product of the reaction being the activated glucosyl donor, ADP glucose. This is utilized by starch synthase for extension of the polysaccharide polymer (reviewed in Hannah 1996).
Initial studies with potato AGP showed that expression in E. coli yielded an enzyme with allosteric and kinetic properties very similar to the native tuber enzyme (Iglesias et al., 1993; Ballicora et al., 1995). Greene et al. (1996a, 1996b) showed the usefulness of the bacterial expression system in their structure-function studies with the potato AGP. Multiple mutations important in mapping allosteric and substrate binding sites have been identified (Okita et al., 1996).
AGP enzymes have been isolated from both bacteria and plants. Bacterial AGP consists of a homotetramer, whereas plant AGP from photosynthetic and non-photosynthetic tissues is a heterotetramer composed of two different subunits. The plant enzyme is encoded by two different genes, with one subunit being larger than the other. This feature has been noted in a number of plants. The AGP subunits in spinach leaf have molecular weights of 54 kDa and 51 kDa, as estimated by SDS-PAGE. Both subunits are immunoreactive with antibody raised against purified AGP from spinach leaves (Copeland et al., 1981; Morell et al., 1988). Immunological analysis using antiserum prepared against the small and large subunits of spinach leaf showed that potato tuber AGP is also encoded by two genes (Okita et al., 1990, supra). The cDNA clones of the two subunits of potato tuber (50 and 51 kDa) have also been isolated and sequenced (Muller-Rober et al., 1990; Nakata et al., 1991). The large subunit of potato tuber AGP is heat stable (Nakata et al., 1991, supra).
As Hannah and Nelson (1975, 1976) postulated, both Shrunken-2 (Sh2) (Bhave et al., 1990) and Brittle-2 (Bt2) (Bae et al., 1990) are structural genes of maize endosperm ADP-glucose pyrophosphorylase. Sh2 and Bt2 encode the large subunit and small subunit Of the enzyme, respectively. Based on cDNA sequencing, Sh2 and Bt2 proteins have predicted molecular weight of 57,179 Da (Shaw et al., 1992) and 52,224 Da, respectively. The endosperm is the site of most starch deposition during kernel development in maize. Sh2 and Bt2 maize endosperm mutants have greatly reduced starch levels corresponding to deficient levels of AGP activity. Mutations of either gene have been shown to reduce AGP activity by about 95% (Tsai and Nelson, 1966, supra; Dickinson and Preiss, 1969, supra). Furthermore, it has been observed that enzymatic activities increase with the dosage of functional wild type (wt) Sh2 and Bt2 alleles, whereas mutant enzymes have altered kinetic properties. AGP is the rate limiting step in starch biosynthesis in plants. Stark et al. (1992) placed a mutant form of E. coli AGP in potato tuber and obtained a 35% increase in starch content.
The cloning and characterization of the genes encoding the AGP enzyme subunits have been reported for various plants. These include Sh2 cDNA (Bhave et al., 1990, supra), Sh2 genomic DNA (Shaw et al., 1992, supra), and Bt2 cDNA (Bae et al., 1990, supra) from maize; small subunit cDNA (Anderson et al., 1989) and genomic DNA (Anderson et al., 1991) from rice; and small and large subunit cDNAs from spinach leaf (Morell et al., 1988, supra) and potato tuber (Muller-Rober et al., 1990, supra; Nakata et al., 1991, supra). In addition, cDNA clones have been isolated from wheat endosperm and leaf tissue (Olive et al., 1989) and Arabidopsis thaliana leaf (Lin et al., 1988). AGP sequences from barley have also been described in Ainsworth et al. (1995).
AGP has been found to function as an allosteric enzyme in all tissues and organisms investigated to date. The allosteric properties of AGP were first shown to be important in E. coli. A glycogen-overproducing E. coli mutant was isolated and the mutation mapped to the structural gene for AGP, designated as glyC. The mutant E. coli, known as glyC-16, was shown to be more sensitive to the activator, fructose 1,6 bisphosphate, and less sensitive to the inhibitor, cAMP (Preiss 1984). Although plant AGP's are also allosteric, they respond to different effector molecules than bacterial AGP's. In plants, 3-phosphoglyceric acid (3-PGA) functions as an activator while phosphate (PO4) serves as an inhibitor (Dickinson and Preiss, 1969, supra).
Using an in vivo mutagenesis system created by the Ac-mediated excision of a Ds transposable element fortuitously located close to a known activator binding site, Giroux et al. (1996) were able to generate site-specific mutants in a functionally important region of maize endosperm AGP. One mutant, Rev6, contained a tyrosine-serine insert in the large subunit of AGP and conditioned a 11-18% increase in seed weight. Published international patent applications WO 99/58698 and WO 98/22601 and issued U.S. Pat. No. 6,069,300 disclose mutations in the large subunit of maize AGP enzyme that, when expressed, confer increased heat stability to the enzyme in comparison to that observed for wild type AGP enzyme. In addition, published international application WO 01/64928 teaches that various characteristics, such as seed number, plant biomass, Harvest Index etc., can be increased in plants transformed with a polynucleotide encoding a large subunit of maize AGP containing the Rev6 mutation.
Ou-Lee and Setter (1985) examined the effects of temperature on the apical or tip regions of maize ears. With elevated temperatures, AGP activity was lower in apical kernels when compared to basal kernels during the time of intense starch deposition. In contrast, in kernels developed at normal temperatures, AGP activity was similar in apical and basal kernels during this period. However, starch synthase activity during this period was not differentially affected in apical and basal kernels. Further, heat-treated apical kernels exhibited an increase in starch synthase activity over control. This was not observed with AGP activity. Singletary et al. (1993, 1994) using an in vitro culture system quantified the effect of various temperatures during the grain fill period. Seed weight decreased steadily as temperature increased from 22-36° C. A role for AGP in yield loss is also supported by work from Duke and Doehlert (1996). These researchers showed that transcript levels decreased to a varying degree, but only one enzyme, AGP, showed a marked decrease in activity with the lower transcript levels. They postulated that AGP may have a faster turnover rate than the other enzymes, and hence is more sensitive to changes in transcript levels. More recent work by Wilhelm et al. (1999) also makes a strong argument for AGP's role in yield loss during heat stress. The Wilhelm et al. authors studied seven inbreds over three replications, and through Q10 analysis, showed that AGP was the only enzyme that exhibited lower activity than the control.
Work by Keeling et al. (1993, supra) quantified SSS activity in maize and wheat using Q10 analysis, and showed that SSS is an important control point in the flux of carbon into starch. In vitro biochemical studies with AGP and SSS clearly show that both enzymes of maize are heat labile. Maize endosperm AGP loses 96% of its activity when heated at 57° C. for five minutes (Hannah et al., 1980). This is in contrast to potato AGP which is fully stable at 70° C. (Sowokinos et al., 1982; Okita et al., 1990). Although the small subunits of AGP are highly conserved among a variety of plant species (Hannah et al., 2001), the N-termini of potato tuber and maize endosperm small subunits exhibit sequence differences. Heat inactivation studies with SSS showed that it is also labile at higher temperatures, and kinetic studies determined that the Km value for amylopectin rose exponentially when temperature increased from 25-45° C. (Jenner et al., 1995, supra).